Probes of rna structure and methods for using the same

ABSTRACT

Methods for obtaining structural data from RNA in a sample, and RNA probes for performing the same, are provided. Methods of reversibly modifying RNA is a sample, in vitro or in vivo, and reversible probes for performing the same, are provided. The RNA probes may be SHAPE probes that include aryl or heteroaryl acyl imidazoles. The RNA probes may be reversible probes that include an aryl or heteroaryl ring substituted with a hydroxyl-reactive group and an azido-containing group. Also provided are methods of comparing in vitro and in vivo RNA structural data. Also provided are methods of diagnosing a cellular proliferative disease condition, e.g., by probing HOTAIR RNA. Aspects of the invention further include compositions, e.g., probes and kits, etc., that find use in methods of the invention.

GOVERNMENT RIGHTS

This invention is made with Government support under grant Nos.R01-GM072705 and R01-HG004361 awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

INTRODUCTION

Despite being composed of only four chemically similar nucleotides, RNAcan base pair with itself and interact with other molecules to formsecondary and tertiary structures. In vitro RNA structure-probing hasimproved the accuracy of secondary structure models and RNA structuralmotifs. The 2′-hydroxyl group is a universal chemical feature in everyRNA. The method of selective 2′-hydroxyl acylation followed by primerextension (SHAPE) has been used to measure and predict the secondarystructures of complex RNAs in in vitro systems. Single-stranded orflexible RNA regions exhibit high 2′-hydroxyl reactivity, whereas RNAnucleotides engaged in base pairing or other interactions show lowerreactivity.

RNA structure in cells is influenced by the rate of transcription, localsolution conditions, the binding of small molecules, and interactionswith numerous RNA-binding proteins. Genomes are extensively transcribedto generate diverse coding and regulatory RNAs which play importantroles in many facets of gene regulation and in diseases such as cancer.However, many RNAs' structures and functions remain to be characterizedin vivo. Probes for elucidating RNA structure and function in cells andin a wide range of organisms to obtain structural maps of RNAs are ofinterest.

SUMMARY OF THE INVENTION

Methods for obtaining structural data from RNA in a sample, and RNAprobes for performing the same, are provided. Methods of reversiblymodifying RNA is a sample, in vitro or in vivo, and reversible probesfor performing the same, are provided. The RNA probes may be SHAPEprobes that include aryl or heteroaryl acyl imidazoles. The RNA probesmay be reversible probes that include an aryl or heteroaryl ringsubstituted with a hydroxyl-reactive group and an azido-containinggroup. Also provided are methods of comparing in vitro and in vivo RNAstructural data. Also provided are methods of diagnosing a cellularproliferative disease condition, e.g., by probing HOTAIR RNA.

In some embodiments the azide functionalized acylation chemicals arefunctionalized after their conjugation to RNA, e.g. using CLICKchemistry to functionalize with a detectable dye, including fluorescentdyes, to a binding partner, e.g. biotin, digoxin, etc., and the like.The functional group can be used for detection or labeling, forisolation or purification of the tagged RNA, and the like.

In some embodiments the compositions, e.g., probes and kits, etc., areprovided that find use in the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict exemplary probes FAI and NAI (A) and the2′-O-acylation of a 2′-hydroxyl group of RNA (B). FIG. 1C shows a timecourse of ATP modification by NAI and FAI. FIG. 1D shows a correlationof 2′-RNA modification of 5S rRNA in vitro using NAI versus N-methylisotoic anhydride (NMIA).

FIG. 2 shows a denaturing gel electrophoresis of NAI modification of 5SrRNA in cells (bottom). Also depicted is secondary structure mapping ofNAI modification of 5S rRNA in cells (top, left), and athree-dimensional model of S.C. 5SrRNA with modifications superimposedonto the structure (PDB 3U5H) (top, right).

FIGS. 3A-3B illustrate that 5S rRNA has different modification patternsin cells: (A) denaturing gel electrophoretic analysis of NAImodification of 5S rRNA in M. musculus Embyronic Stem cells and invitro; and (B) normalized Differential profile of M. musculus EmbyronicStem cell 5S rRNA.

FIGS. 4A-4D: A. Three-dimensional model of S.C. 5SrRNA with differentialmodifications superimposed onto the structure (PDB 3U5H). (B) Closeupview of hyperreactivity of M.M. A49 and S.C. U50. U50 is near the kinkof helix II and helix III, This conformation, which allows Loop C tointeract with 28S rRNA promotes the ejection of S.C. U50 2′-OH thusrendering it more dynamic and reactive to NAI modification. (C)Zoomed-in view of a three-nucleotide bridge that joins loop A to helixII. This conformation results in docking of A11, hiding its 2′-OHthrough a hydrogen bond with A13-OP2. In addition C10 is forced into astacking interaction with PHE20, which exposes the 2′-OH, potentiallyincreasing its reactivity with NAI. (D). Close up view of differentialmodification of Helix IV. The ribosome crystal structure shows many ofthe residues that are hypomodified in cells are engaged in extensiveinteractions with ribosomal RNA. Shown is the interaction of U86 with28S rRNA. Such extensive bonding and stacking may be stabilizing theinternucleotide linkages of these residues, limiting their acylationreactivity.

FIGS. 5A-5C: (A) Three-dimensional model of S.C. 5SrRNA colored byb-factor (PDB 3U5H). (B) Denaturing gel electrophoretic analysis of NAImodification of 5S rRNA in S. cerevisiae cells and in vitro. (C)Normalized differential profile of S. cerevisiae 5S rRNA. Residues arelabeled for their importance in 5S rRNA function as noted in Smith etal., Saturation mutagenesis of 5S rRNA in Saccharomyces cerevisiae. MolCell Biol 21, 8264-8275, (2001).

FIGS. 6A-6C. Gel shift acylation reactions of NAI and FAI probes: (A)reaction with ATP versus dATP; (B-C) concentration and time coursestudies with FAI (B); and NAI (C).

FIG. 7 illustrates the gel shift results of quenching of acylationreactions of NAI and FAI with ATP using β-mercaptoethanol (B-Me).

FIGS. 8A-8H. Characterization of probe reactivity with 5S RNA. (A) Gelelectrophoresis comparing reactivities of NMIA, FAI, and NAI. (B)Secondary structure map to Mus. Musculus 5S rRNA. (C) Normalized SHAPEreactivity for NMIA from (A). (D) Normalized SHAPE reactivity for FAIfrom (A). (E) Normalized SHAPE reactivity for NAI from (A). (F)Correlation of position-dependent reactivities of NMIA and NAI. (G)Comparison between NMIA and FAI. (H) Comparison between NAI and FAI.

FIGS. 9A-9B illustrate gel shift results of probe reactivity with RNA incells: (A) Increasing amounts of NAI yield concentration-dependentreverse transcription stops; and (B) Higher concentrations of NAI andFAI produce reverse transcription stops.

FIG. 10: Bright field imaging of V6.5 mESCs. Images show little evidenceof membrane and cell morphology disruption during one hour whenincubated with NAI or DMSO vehicle control as compared to PBS (TOP ROW).Even after 60 minutes of NAI treatment, ESCs remained attached to tissueculture vessel, appeared morphologically normal and unstained by trypanblue (BOTTOM ROW).

FIGS. 11A-11F: NAI modifies nuclear, lower abundant RNAs. (A) Denaturinggel electrophoresis or U1 snRNA RT products. (B) Denaturing gelelectrophoresis of U2 snRNA RT products. (C) Denaturing gelelectrophoresis of SNORD3A RT products. (D) Secondary structure mappingof U1 snRNA RT products. (E) Secondary structure mapping of U2 snRNA RTproducts. (F) Secondary structure mapping of SNORD3A snRNA RT products.Secondary structures are represented by their predicted in vitro folds,not those in their RNP complexes.

FIGS. 12A-12D illustrate the results of acylation reactions using NAIwith denaturing gel electrophoresis: (A) Homo sapiens MDA-MB-231 cells5SrRNA RT products; (B) Saccharomyces cerevisiae cells 5S rRNA RTproducts; (C) Escherichia coli cells 5S rRNA RT products; (D) Drosophilamelanogaster cells 5S rRNA RT products.

FIGS. 13A-13D illustrate the mechanism of action (A), synthesis (B) andevaluation (C-D) of a reversible acylation probe.

FIGS. 14A-14B illustrate the results of secondary structure mapping ofHOTAIR RNA using the reversible RNA probe ABI-1: (A) Gel electrophoresisof cDNA products; and (B) Correlation between 2′-OH reactivity to NMIAand ABI-1.

FIGS. 15A-15C illustrate reversible acylation of RNA and cellpermeability of reversible acylation probes: (A) Gel electrophoresis ofcDNA products using ABI-1, with and without triphenylphosphine (TPP);(B) Chemical structures of ABI-1, NAI, and NAI-1; (C) structuralmodification of RNA in various cells using NAI-1.

FIGS. 16A-16E. NAI-1 was used for conjugation to fluorophores andenrichment handles using “click” chemistry. (A, B) Using theazide-linked acylation reagent fluorescent dye was linked to NAI-1/RNAcomplexes. (C) Hydroxyls from ATP are acylated. (d) The acylated ATP issupershifted with copper free “click” chemistry with DIBO-biotin. (E)Enrichment for biotin labeled RNAs using a streptavidin pulldown inwhich the isolated RNAs are eluted then probed using a streptavidindot-blot.

FIGS. 17A-17B. Functionalized RNA can be enriched to decreasebackground. (A) Purified RNAs that map back to segments of RNA predictedto be acylated were enriched by functionalizing with biotin throughcopper-free “click” chemistry, and selection for the biotin tag.

DEFINITIONS

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As used herein, “suitable conditions” for carrying out a synthetic stepare explicitly provided herein or may be discerned by reference topublications directed to methods used in synthetic organic chemistry.The reference books and treatise set forth above that detail thesynthesis of reactants useful in the preparation of compounds of thepresent invention, will also provide suitable conditions for carryingout a synthetic step according to the present invention.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution. The term lower alkyl will be used herein as known in theart to refer to an alkyl, straight, branched or cyclic, of from about 1to 6 carbons.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—. Use of a single dash (“—”) or double dash (“—” or “--”) refersto a single covalent bond, while use of “═” refers to a double bond. Thesymbol, )₂ or ₂(, when displayed with —S, indicates that the compoundinside the parenthesis may be present as a dimer forming a disulfidebond. The dimer may be reduced to a monomer.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof, havingthe stated number of carbon atoms and an acyl radical on at least oneterminus of the alkane radical. The “acyl radical” is the group derivedfrom a carboxylic acid by removing the —OH moiety therefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl”, where “heteroalkyl” refers to carbon chains having one ormore substitutions at one or more carbon atoms of the hydrocarbon chainfragment. Alkyl groups that are limited to hydrocarbon groups are termed“homoalkyl”. Certain alkyl groups include those containing between aboutone and about twenty five carbon atoms (e.g. methyl, ethyl and thelike).

The term “lower alkyl” generally refers to a straight, branched, orcyclic hydrocarbon chain containing 8 or fewer carbon atoms, and cancontain from 1 to 8, from 1 to 6, or from 1 to 4 carbon atoms. Certain“lower alkyl” groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and thelike. “Lower alkyls” can be optionally substituted at one or more carbonatoms of the hydrocarbon chain.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused to refer to those alkyl groups attached to the remainder of themolecule via an oxygen atom, an amino group, or a sulfur atom,respectively.

By “heteroatom” is meant atoms other than a carbon which may be presentin a carbon backbone or a linear, branched or cyclic compound. Certainheteroatoms include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P)and silicon (Si). Heteroatoms can be present in their reduced forms,e.g., —OH, —NH, and —SH.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, having the statednumber of carbon atoms and at least one heteroatom which can be a memberselected from O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom canoptionally be quaternized. Normally heteroalkyl groups contain no morethan two heteroatoms linked in sequence. The heteroatom(s) O, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Generally, up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (usuallyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms which are members selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Certain substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, =NR′, =N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)=NR″“,—NR—C(NR′R”)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ where eachcan be independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of theembodiments includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, =NR′, =N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′“, —NR″C(O)₂R′, —NR—C(NR′R″R”′)=NR″“,—NR—C(NR′R”)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ can beindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the embodiments includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

The term “amino” or “amine group” refers to the group —NR′R″ (orN±FIR′R″) where R, R′ and R″ are independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl,substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine is an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —N⁺RR′R″ and itsbiologically compatible anionic counterions.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, geometric isomers, individual isomers andother stereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as, their racemic and optically pure forms. Optically active (+)and (−), (R)- and (S)-, or (D)- and (L)-isomers may be prepared usingchiral synthons or chiral reagents, or resolved using conventionaltechniques, such as reverse phase HPLC. When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

In some embodiments the RNA modification provides a reactant group forCLICK chemistry reactions (see Click Chemistry: Diverse ChemicalFunction from a Few Good Reactions Hartmuth C. Kolb, M. G. Finn, K.Barry Sharpless Angewandte Chemie International Edition Volume 40, 2001,P. 2004, herein specifically incorporated by reference).

DETAILED DESCRIPTION OF THE EMBODIMENTS

As summarized above, methods for obtaining structural data from RNA in asample, and RNA probes for performing the same, are provided. Methods ofreversibly modifying RNA is a sample, in vitro or in vivo, andreversible probes for performing the same, are also provided. Thesubject RNA probes may be SHAPE probes that include an aryl orheteroaryl ring substituted with an acyl imidazole and a modulatingsubstituent. The subject RNA probes may be reversible probes thatinclude an aryl or heteroaryl ring substituted with a hydroxyl-reactivegroup and an azido-containing group.

RNA Probes

As summarized above, aspects of the invention include probes of RNAstructure. In general terms, the subject RNA probes include ahydroxyl-reactive functional group attached to an aryl or heteroarylring where the ring may be further substituted at an adjacent positionof the ring with a modulating substituent. The hydroxyl-reactive groupis capable of reacting with one of more unconstrained nucleotides of aRNA to produce a 2′-modified RNA. The modulating substituent, ifpresent, modulates the reactivity of the hydroxyl-reactive group. Themodulating substituent may be selected to tune the reactivity of theprobe to provide a desired reactivity with the 2′-hydroxyl groups ofRNA, and a desired stability under physiological conditions, e.g., asmeasured by half-life and/or minimum incubation times.

Any suitable aryl or heteroaryl ring may be utilized in the subjectprobes. Aryl and heteroaryl rings of interest include, but are notlimited to, phenyl, pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl,imidazolyl, oxazolyl, pyrimidinyl, pyrazinyl and pyridazinyl rings. Insome instances, the aryl or heteroaryl ring further comprises one ormore groups independently selected from a solubility-enhancing group, abinding moiety, a tag, a detectable label, a permeability-enhancinggroup and a functional group (e.g., a bioorthogonal group for attachinga detectable label (e.g., a fluorophore) or a solid support). Suchgroups may be included as part of the ring or in a substituent of thering. Chemical modifications can be made to the probe to enhanceappropriate solubility. In some cases, the solubility-enhancing group isa basic group that may be protonated and charged under physiologicalconditions. In other cases, the solubility-enhancing group is a polargroup (e.g., a heteroatom containing ring or substituent) that isneutral under physiological conditions but which is hydrophilic. Incertain instances, the solubility-enhancing group is a basic center(e.g., a N atom) that is part of the heteroaryl ring (e.g., a pyridylring). By solubility-enhancing is meant that the solubility of a probeat physiological conditions is increased by the inclusion of the groupof interest, relative to a corresponding probe that lacks thesolubility-enhancing group. For example, a pyridyl ring has enhancedsolubility at physiological pH over a corresponding benzyl ring becauseit includes a basic nitrogen in the ring. Chemical modifications can bemade to the probe to enhance appropriate cell permeability by, e.g., (1)changing the pKa of the probe, (2) adding an ionicpermeability-enhancing group, either cationic or anionic, attachedthrough an appropriate linker to the aryl or heteroaryl ring, or (3)adding nonionic permeability-enhancing groups such as ethylene glycol orpolyethylene glycol moieties.

Any suitable hydroxyl-reactive groups may be utilized in the subjectprobes. Hydroxyl reactive groups of interest include, but are notlimited to, active esters, epoxides, oxiranes, oxidizing agents,aldehydes, alkyl halides (e.g., benzyl halides), isocyanates, and othergroups such as those described by Hermanson, Bioconjugate Techniques,Second Edition, Academic Press, 2008. In some cases, thehydroxyl-reactive group is an active ester. Any convenient active estergroup may be utilized in the subject RNA probes. The active ester of theprobe may react with one of more uncontrained 2′-hydroxyl groups of anRNA to produce a 2′-acylated RNA. In some instances, the active ester isan acyl imidazole. The acyl imidazole (—C(O)-imidazolyl) group mayinclude an imidazolyl that is further substituted with one or moresubstituents, including but not limited to, a lower alkyl (e.g., amethyl or ethyl group), a halo (e.g. a bromo, a chloro or a fluoro) andnitro. In certain instances, the imidazolyl group is a2-methyl-imidazolyl or a 4-methyl-imidazolyl.

Any suitable modulating substituent may be included at the 2-position ofthe aryl or heteroaryl ring (2-position relative to thehydroxyl-reactive group substituent). By “adjacent position of the ring”is meant that that two ring substituents (e.g., the hydroxyl-reactivegroup and the modulating substituent) are attached to the ring atconsecutive ring positions, i.e., at positions 1- and 2- of a ring,relative to each other. In some instances, the hydroxyl reactive groupand modulating substituent are selected to provide for a desired probereactivity and stability. In some embodiments, the hydroxyl reactivegroup is acyl imidazole, and the modulating substituent is an alkylgroup. The proximity of the modulating substituent, at the adjacentposition on the ring to the acyl imidazole, allows the modulatingsubstituent to modulate the reactivity of the acyl imidazole group.Without wishing to be bound by theory, in some cases the modulatingsubstituent may have a steric effect on the acyl imidazole group. Incertain instances, the modulating substituent is an alkyl group such asa lower alkyl group, optionally further substituted with one or moresubstituents.

In some embodiments, the hydroxyl reactive group and the modulatingsubstituent are selected to provide a RNA probe with a desired half-lifein the sample, e.g., a half-life of 5 minutes or more, such as 10minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes ormore, 50 minutes or more, 60 minutes or more, 2 hours or more, 3 hoursor more, or even 6 hours or more.

In some instances, the modulating substituent is further substitutedwith a masked group. As used herein, the term “masked group” refers to agroup of the probe that can be selectively unmasked to produce anunmasked reactive functional group. A masked group is stable, e.g., tophysiological conditions, until it is contacted with a stimulus capableof unmasking the masked group. As such, in some instances, the probe mayinclude a masked group that is unmasked upon application of a stimulus.Any suitable groups may be utilized as a masked group in the subjectprobes. Groups of interest include, but are not limited to, protectedfunctional groups (e.g., protected nucleophilic groups), convertiblefunctional groups and bioorthogonal groups. As used herein “convertiblefunctional group” refers to a stable functional group that uponapplication of a suitable stimulus is transformed into a reactivefunctional group, e.g., a functional group that is capable of reactingspontaneously. As such, unmasking of a masked group may includedeprotection, or alternatively, conversion of a stable functional groupto a reactive functional group (e.g., a nucleophilic group). Theunmasked group may then react spontaneously (e.g., intramolecularly atan adjacent electrophilic center) to reverse modification of the RNA. Insome cases, unmasking a masked group includes deprotecting. In certainembodiments, the RNA probe does not include a modulating substituent atthe 2-position relative to the hydroxyl-reactive group.

In some embodiments, the RNA probe of the invention is described byformula (I):

-   -   where Y is a hydroxyl-reactive group, A is an aryl or a        heteroaryl ring, and R¹ is H or a lower alkyl.

One aspect of the invention is a SHAPE probe that acylates one or more2′-hydroxyl groups of a RNA of interest in a sample. As such, in formula(I), Y may include an active ester functional group. Any convenientactive ester group may be utilized in the subject SHAPE probes. Theactive ester of the probe may react with one of more unconstrainednucleotides of a RNA to produce a 2′-acylated RNA. In some instances,the active ester is an acyl imidazole. The acyl imidazole(—C(O)-imidazolyl) group may include an imidazolyl that is furthersubstituted with one or more substituents, including but not limited to,a lower alkyl (e.g., a methyl or ethyl group), a halo (e.g. a bromo, achloro or a fluoro) and a nitro. In certain instances, the imidazolylgroup is a 2-methyl-imidazolyl or a 4-methyl-imidazolyl.

Any convenient aryl or a heteroaryl ring may be utilized in the subjectSHAPE probes. In addition to an active ester substituent (e.g., the acylimidazole group of formula (I)), the aryl or heteroaryl ring may befurther substituted, e.g., with a modifying substituent (R¹) at the2-position relative to the active ester.

In some embodiments, a SHAPE probe of the invention is described byformula (II):

-   -   wherein A is an aryl or a heteroaryl ring; R¹ is H or a lower        alkyl; and R² is H, a lower alkyl, a halo or nitro.

In certain instances, in formula (II), A is further substituted at anyconvenient position, with a functional group that is bioorthogonal,i.e., a functional group that is stable under the physiologicalconditions of a sample of interest, but which selectively reacts with acomplementary reagent (e.g., a detectable label reagent). A variety ofbioorthogonal chemistries and reagents may be utilized in the subjectprobes and reagents, including but not limited to, Staudinger ligations(e.g., using azido and phosphine groups), copper-free click chemistry(e.g., using azido and cyclooctyne groups), oxime or hydrazine chemistry(using aldehydes and ketones). The introduction of such a functionalgroup as a substituent of the aryl or heteroaryl ring (A) allows for the2′-acylated RNA to be further modified with a moiety such as adetectable label (e.g., a fluorophore or a peptide tag), or a solidsupport.

In some embodiments, the SHAPE probe is described by one of formulas(III) and (IV):

where R¹ and R² are as defined above, Z¹, Z², Z³, Z⁴ Z⁵, Z⁶ and Z⁷ areindependently selected from O, S, CR³, N and NR³, and where R³ is H oran aryl group substituent.

In some cases, in formulas (I) to (IV), the probe includes an aryl orheteroaryl ring (A) selected from a phenyl, a pyridyl, a pyrrolyl, afuranyl, a thienyl, a thiazolyl, an imidazolyl, an oxazolyl, apyrimidinyl, a pyrazinyl and a pyridazinyl ring.

In certain embodiments, the SHAPE probe is described by one of thefollowing structures:

where R¹ is as defined above, Z⁸ is O, S or NR⁴, wherein R⁴ is H or alower alkyl; and where the imidazolyl group of the active ester isoptionally substituted with a lower alkyl, a halo or nitro.

In certain embodiments, R¹ is a lower alkyl (e.g., a methyl, an ethyl, apropyl, an isopropyl, a butyl, or a tert-butyl). R¹ may be furthersubstituted with azido, hydroxyl, thiol, an amino, or a protectedversion thereof.

In certain embodiments, R¹ is selected from one of the following groups:

where X is selected from —N₃, hydroxyl, protected hydroxyl, thiol,protected thiol, amino and protected amino; and R⁵ is H or an alkylgroup substituent. In some instances, R⁵ is H, a lower alkyl, hydroxyl,or an alkyloxy. In certain instances, R¹ is —CH₂—N₃ or —CH₂CH₂N₃.

As summarized above, another aspect of the invention is a reversible RNAprobe. In some instances, the probe includes a modulating substituent(e.g., R¹) that includes a masked group (e.g., as described herein) suchthat the probe reversibly modifies RNA. The modification of one or moreunconstrained nucleotides of the 2′-modified RNA by the probe may bereversed by the application of a stimulus. Application of the stimulusto the sample unmasks a masked group of the probe which leads tocleavage of the probe from the 2′-modified RNA. In some cases, the2′-modified RNA is 2′-acylated RNA and application of the stimulus tothe sample may be described as de-acylating the 2′-acylated RNA.

By “reversibly modifying” or “reverse modification” is meant that themodification of a RNA in a sample may be reversed upon application of asuitable stimulus that unmasks a masked group of the probe. By“reversibly acylates” is meant that the acylation of a RNA of interestin a sample may be reversed upon application of a suitable stimulus(e.g., a photon, a deprotection reagent or a chemical agent) thatunmasks a masked reactive group of the probe. In some cases, theunmasked reactive group may then react intra-molecularly at the adjacent2′-acyl group to release 2′-hydroxyl RNA.

In certain embodiments, the masked group is an azido group. The azidogroup may be “unmasked” by reaction with a reagent such as a phosphineor a dithiol. Without wishing to be bound by theory, reaction of theazido group with a phosphine reagent may lead to an iminophosphorane(aza-ylide) intermediate, which can react intramolecularly with anadjacent 2′-acyl electrophilic group to produce a covalent amide bond(see e.g., FIG. 13A).

In some embodiments, the masked group is a functional group protected bya photolabile protecting group. In such cases, the stimulus may be aphoton and application of the stimulus photocleaves the masked group toproduce a reactive functional group.

In some cases, the reversible probe includes a hydroxyl-reactive groupand an azido group linked by an aryl or heteroaryl ring. In someinstances, the azido group is included as part of an alkyl substituent(e.g., a C₁-C₆ alkyl substituent that may be branched or straightchained) of the aryl or heteroaryl ring (e.g., the modulatingsubstituent). In such cases, the azido group is not directly attached tothe ring but is attached to a carbon of the substituent that is one ormore atoms away from the ring, such as 1, 2 or 3 or more atoms removedfrom the ring. In other instances, the azido group is directly attachedto the ring.

In some cases, the hydroxyl-reactive group and the azido group areattached to the aryl or heteroaryl ring at neighboring positions of thering (e.g., at the 1- and 2-positions of a ring). In some embodiments,the probe is described by formula (V):

where Y is a hydroxyl-reactive group; A is an aryl or a heteroaryl ring;and L is a lower alkyl group, optionally substituted with an alkyl groupsubstituent. In certain embodiments, Y is an active ester.

In certain embodiments, A comprises one or more groups independentlyselected from a solubility-enhancing group, a binding moiety, apermeability-enhancing group, a detectable label and a bioorthogonalgroup (e.g., for attaching a detectable label (e.g., a fluorophore) or asolid support).

In certain instances, in formula (V), A is further substituted at anyconvenient position, with a functional group that is bioorthogonal,i.e., a functional group that is stable under the physiologicalconditions of a sample of interest, but which selectively reacts with acomplementary reagent (e.g., a detectable label reagent). A variety ofbioorthogonal chemistries and reagents may be utilized in the subjectprobes and reagents, including but not limited to, Staudinger ligations(e.g., using azido and phosphine groups), copper-free click chemistry(e.g., using azido and cyclooctyne groups), oxime or hydrazine chemistry(using aldehydes and ketones). The introduction of such a functionalgroup as a substituent of the aryl or heteroaryl ring (A) allows for the2′-modified RNA to be further modified with a moiety such as adetectable label (e.g., a fluorophore or a peptide tag), or a solidsupport.

In certain embodiments, the reversible probe is described by one offormulas (VI) and (VII):

where: Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are independently selected from O,S, CR, N and NR, where R is H or an aryl group substituent; B and D arearyl or heteroaryl rings; and X is halo, imidazolyl, anN-hydroxylsuccinimidyl (e.g., NHS or sulfo-NHS), an alkoxy (e.g.,methoxy) or an aryloxy (e.g., pentafluorophenyloxy); and m is 1 or 2. Informula (VI), Z¹, Z², Z³ and Z⁴ are selected to provide an aryl orheteroaryl ring. In formula (VII), Z⁵, Z⁶ and Z⁷ are selected to providean aryl or heteroaryl ring. In certain embodiments, in formulas (VI) and(VII), the B and D rings are aryl or heteroaryl rings selected from aphenyl, a pyridyl, a pyrrolyl, a furanyl, a thienyl, a thiazolyl, animidazolyl, an oxazolyl, a pyrimidinyl, a pyrazinyl and a pyridazinylring.

In some cases, the reversible probe is described by one of the followingstructures:

where Z⁸ is O, S or NR⁴, wherein R⁴ is H or a lower alkyl; X is asdefined above, and m is 1 or 2.

In certain embodiments, X is an imidazolyl, optionally substituted witha lower alkyl (e.g., a 2-methyl or a 4-methyl), a halo (e.g., a bromo,chloro or fluoro) or nitro. In certain embodiments, m is 1.

Masked Groups

As used herein, the term “masked group” refers to a group of the probethat can be selectively unmasked to produce an unmasked reactivefunctional group. The masked group may be unmasked after contact with astimulus (e.g., light, or a chemical agent) under suitable conditions.The modifiable group is capable of modification under conditions atwhich target molecules of interest are able to be maintained in a nativestate in a sample (e.g., physiological conditions at which RNA structureis maintained in a cell). The masked group may be unmasked to produce anunmasked group that is capable of spontaneous reaction with an adjacentcompatible functional group (e.g., intramolecular or intermolecular).

The masked group may be reactive with the functional group of a chemicalagent (e.g., an azido-containing masked group that is reactive with aphosphine reagent or a dithiol). A variety of functional groupchemistries and chemical agent stimuli suitable for unmasked them may beutilized in the subject probes and methods. Functional group chemistriesand chemical agents of interest include, but are not limited to, Clickchemistry groups and reagents (e.g., as described by Sharpless et al.,(2001), “Click Chemistry: Diverse Chemical Function from a Few GoodReactions”, Angewandte Chemie International Edition 40 (11): 2004-2021),Staudinger ligation groups and reagents (e.g., as described by Bertozziet al., (2000), “Cell Surface Engineering by a Modified StaudingerReaction”, Science 287 (5460): 2007), and other bioconjugation groupsand reagents (e.g., as described by Hermanson, Bioconjugate Techniques,Second Edition, Academic Press, 2008). In certain embodiments, themodifiable group includes a functional group selected from an azido, aphosphine (e.g., a triaryl phosphine or a trialkyl phosphine or mixturesthereof), a dithiol, an active ester, an alkynyl, a protected amino, aprotected hydroxy, a protected thiol, a hydrazine, and a disulfide.

In some instances, the masked group includes an azido group, such asthose contained in the azido linkers described in US2001/0014611.

The masked group may be cleavable, e.g., include a cleavable bond. Asused herein, the term “cleavable” refers to a moiety that includes acleavable covalent bond that can be selectively cleaved to produce twoproducts. Application of a suitable cleavage stimulus to a probe thatcontains a cleavable bond will produce two products. As used herein, theterm “cleavage conditions” refers to the conditions in which a cleavablebond may be selectively cleaved. Irradiation of a sample with light of asuitable wavelength that is absorbed by a photocleavable group is anexample of a cleavage condition. A variety of cleavable protectinggroups, linkers and functional groups are known to those of skill in theart and find use in the subject probes, e.g., as described in Olejnik etal. (Methods in Enzymology 1998 291:135-154), and further described inU.S. Pat. No. 6,027,890; Olejnik et al. (Proc. Natl. Acad Sci,92:7590-94); Ogata et al. (Anal. Chem. 2002 74:4702-4708); Bai et al.(Nucl. Acids Res. 2004 32:535-541); Zhao et al. (Anal. Chem. 200274:4259-4268); and Sanford et al. (Chem. Mater. 1998 10:1510-20).Cleavable groups and linkers including the same that may be employed inthe subject probes include electrophilically cleavable groups,enzymatically cleavable groups, nucleophilically cleavable groups,photocleavable groups, metal cleavable groups,electrolytically-cleavable groups, and groups that are cleavable underreductive and oxidative conditions. A cleavable group or linker may beselectively cleaved without breaking other cleavable bonds in themolecule.

The masked group may be photoreactive (e.g., reactive with a stimulussuch as a photon or light of a particular wavelength). In someinstances, the photoreactive group is photocleavable, photoisomerizable(or photoswitchable), or photoactivateable.

In certain embodiments, the masked group includes a photocleavablegroup, where application of a suitable light stimulus activates thegroup and leads to intramolecular cleavage of the 2′-modified RNA. Anyconvenient photocleavable groups may find use in the subject probes.Cleavable groups and linkers may include photocleavable groupscomprising covalent bonds that break upon exposure to light of a certainwavelength. Suitable photocleavable groups and linkers for use in thesubject probes include ortho-nitrobenzyl-based linkers, phenacyllinkers, alkoxybenzoin linkers, chromium arene complex linkers,NpSSMpact linkers and pivaloylglycol linkers, as described in Guillieret al. (Chem. Rev. 2000 1000:2091-2157). For example, a1-(2-nitrophenyl)ethyl-based photocleavable linker (Ambergen) can beefficiently cleaved using near-UV light, e.g., in >90% yield in 5-10minutes using a 365 nm peak lamp at 1-5 mW/cm². In some embodiments, themasked group is a photocleavable group such as a nitro-aryl group, e.g.,a nitro-indole group or a nitro-benzyl group, including but not limitedto: 2-nitroveratryloxycarbonyl, α-carboxy-2-nitrobenzyl,1-(2-nitrophenyl)ethyl, 1-(4,5-dimethoxy-2-nitrophenyl)ethyl and5-carboxymethoxy-2-nitrobenzyl. Nitro-indole groups of interest include,e.g., a 3-nitro-indole, a 4-nitro indole, a 5-nitro indole, a6-nitro-indole or a 7-nitro-indole group, where the indole ring may befurther substituted at any suitable position, e.g., with a methyl groupor a halo group (e.g., a bromo or chloro), e.g., at the 3-, 5- or7-position. In certain embodiments, the nitro-aryl group is a 7-nitroindolyl group.

In some embodiments, the masked group is acid or base labile, e.g.,cleavable with an acidic or a basic reagent. In another embodiment, themasked group is pH sensitive, such that application of a stimulus suchas a suitable pH condition (e.g., a low pH condition below theisoelectric point of the group) modifies the group, e.g., by changing aneutral group (e.g., an amino or carboxylic acid group) into a chargedgroup (e.g., an ammonium or a carboxylate group). In certainembodiments, the masked group is an acid/base labile group of thestructure: —NHC(O)OR³ where R³ is selected from a methyl, ethyl,methoxymethyl, CH₂CH₂F, methylthiomethyl, β-glucuronide,β-galacturonide, D-glucopyranosyl, β-D-galactopyranosyl,tetra-O-acetyl-D-glucopyranosyl, and atetra-O-acetyl-β-D-galactopyranosyl group. In certain instances, theacid labile group is peptide sequence susceptible to cleavage at a pHbetween pH1 and pH4 (e.g., pH 2-4 or pH 3-4). In certain embodiments,the MCIP includes an acid-cleavable linker as described inUS2012/0122153, such as a linker comprising a peptide selected from thegroup consisting of: (SEQ ID NO: 1) DPDP, (SEQ ID NO: 2) DPDPDP, (SEQ IDNO: 3) DPDPDPDP, (SEQ ID NO: 4) DPDPDPP, (SEQ ID NO: 5) DPDPPDPP, (SEQID NO: 6) DPDPPDP, and (SEQ ID NO: 7) DPPDPPDP. In other instances, theacid labile group is a pH sensitive hydrazones (see e.g., BioconjugateChem., 2010, 21 (1), pp 5-13 and Clin. Cancer Res. 2005 11(2 Pt1):843-52).

In some embodiments, the masked group is a protected hydroxyl group,such as a silyl ether group (e.g., —OSiR₃). The silyl ether may beunmasked by application of a stimulus such as a chemical agent (e.g., afluoride reagent) that cleaves the silyl ether and leads to anintramolecular reaction with an adjacent acyl electrophilic center torelease 2′-RNA. An exemplary masked group, probe and stimulus isillustrated in the following scheme:

where X is N or CH, and R is RNA.

In some embodiments, the azido-containing masked group is anazido-substituted alkyl ether (e.g., an azido-methylether). Theazido-substituted alkyl ether may be unmasked by application of astimulus such as an azido reactive chemical agent (e.g., a phosphine ordithiol reagent) that converts the azido group to a reactive group(e.g., an amino group) via a Staudinger-type reaction. Without wishingto be bound by theory, unmasking of an azido-methylether group mayproduce an unstable functional group (e.g., an alpha-amino ether) thatspontaneously hydrolyses and leads to intramolecular reaction with anadjacent acyl electrophilic center to release 2′-RNA. An exemplarymasked group, probe and stimulus is illustrated in the following scheme:

where X is N or CH, and R is RNA.

In some embodiments, the masked group is a protected hydroxyl group thatmay be used in conjunction with an adjacent hydroxyl reactive group thatis trimethyl lock derivative (e.g., an o-hydroxydihydrocinnamic acidderivative). Deprotection of the protected hydroxyl group leads tolactonization with the adjacent electrophilic acyl group to release2′-RNA. Any convenient trimethyl lock compound may be adapted for use inthe subject probes, such as those described by Raines et al. “Trimethyllock: a trigger for molecular release in chemistry, biology, andpharmacology”, Chem. Sci., 2012, 3, 2412-2420, the disclosure of whichis herein incorporated by reference. An exemplary masked group, probeand stimulus is illustrated in the following scheme:

where PG is a hydroxyl protecting group and R is RNA.

Methods

As summarized above, aspects of the invention include methods forobtaining structural data from a RNA in a sample. Further aspects of theinvention include methods for reversibly modifying RNA in a sample. Thesubject methods include modifying one or more unconstrained nucleotidesof the RNA to produce a 2′-modified RNA. In some cases, modification ofthe RNA includes acylation to produce a 2′-acylated RNA. In other cases,modification does not include acylation, e.g, when using probesincluding hydroxyl-reactive groups that are not active esters (e.g., asdescribed herein). In such cases, modification may include an alkylationor addition reaction of a 2′-hydroxy of the RNA of interest.

As such, aspects of the method include contacting the sample with a RNAprobe under conditions by which one or more unconstrained nucleotides ofthe RNA are modified by the probe to produce a 2′-modified RNA. Anyconvenient protocol for contacting the sample with the probe may beemployed. The particular protocol that is employed may vary, e.g.,depending on whether the sample is in vitro or in vivo. For in vitroprotocols, contact of the sample with the probe may be achieved usingany convenient protocol. In some instances, the sample includes cellsthat are maintained in a suitable culture medium, and the probe isintroduced into the culture medium. For in vivo protocols, anyconvenient administration protocol may be employed. Depending upon thereactivity of the probe, the RNA of interest, the manner ofadministration, the half-life, the number of cells present, variousprotocols may be employed. The term “sample” as used herein relates to amaterial or mixture of materials, typically, although not necessarily,in fluid form, containing one or more components of interest.

The subject methods may further include evaluating the samplemodification of the RNA. Evaluation of the sample may be performed usingany convenient method, and at any convenient time. Evaluation of thesample may be performed continuously, or by sampling at one or more timepoints during the subject method. In some embodiments, the evaluatingstep is performed prior to obtaining structural data. In certain cases,evaluation is performed using a cell-based assay that measures theoccurrence of a biological event triggered by the RNA. In other cases,evaluation is performed in conjunction with obtaining structural data.Any observable biological property of interest may be used in theevaluating step of the subject methods.

The subject methods may further include analyzing the 2′-acylated RNA inthe sample to obtain structural data. Selective 2′-hydroxyl acylationfollowed by primer extension (SHAPE) is a method for obtainingstructural data from a RNA. Any suitable SHAPE methods and reagents maybe utilized in practicing the subject methods. SHAPE methods andreagents of interest include, but are not limited to, those described byWeeks and Mauger, “Exploring RNA Structural Codes With SHAPE Chemistry”,Accounts of Chemical Research, 2011, 44 (12), 1280-1291; and Weeks etal. US 2010/0035761, the disclosures of which are herein incorporated byreference.

Once the RNA of interest has been 2′-modified in a sample (e.g., asevaluated by the occurrence of a particular biological event), themodification may be maintained for a period of time, and/or may bereversed via application of a stimulus to the sample.

Aspects of the methods include applying a suitable stimulus to reversemodification of the 2′-modified RNA. Any suitable stimulus may beutilized to reverse modification of the 2′-modified RNA by a reversibleprobe of the invention. In some instances, the stimulus is a photon. Inother instances, the stimulus is a chemical agent (e.g., a deprotectionagent or an azido-reactive agent).

In some instances, the stimulus is a deprotection reagent andapplication of the stimulus deprotects a protected functional group ofthe probe, such as a protected amino, hydroxyl or thiol. In otherinstances, the stimulus is an azido-reactive reagent, and application ofthe stimulus modifies an azido group of the probe, e.g., via aStaudinger-type reaction. Any suitable azido-reactive reagents may beutilized as a stimulus. Azido-reactive reagents of interest include, butare not limited to, dithiols and phosphines such as, arylphosphines(e.g., a triphenyl phosphine), alkylphosphines (e.g., atrialkylphosphine such as tris(2-carboxyethyl)phosphine (TCEP)), orarylalkylphosphines.

In certain embodiments, the stimulus is application of a chemical agent,where the chemical agent is bound to a solid support. Any convenientsupports and methods may be utilized, including but not limited to,chromatographic supports and methods, arrays, beads, etc.

Application of a chemical agent stimulus can be achieved using anyconvenient method, including contacting the sample with the chemicalagent using any convenient method. The particular protocol that isemployed may vary, e.g., depending on whether the sample is in vitro orin vivo. For in vitro protocols, contact of the chemical agent with thesample may be achieved using any convenient protocol. In some cases, asolution of the chemical agent is added to the sample to provide a finalconcentration of the chemical agent in the sample sufficient to modifythe RNA of interest. In some instances, the sample includes cells thatare maintained in a suitable culture medium, and the chemical agent isintroduced into the culture medium. For in vivo protocols, anyconvenient administration protocol may be employed. Depending upon thereactivity of the chemical agent, the response desired, the manner ofadministration, the half-life or stability of the chemical agent, thenumber of cells present, various protocols may be employed.

In some embodiments, the stimulus is a photon. Any suitable source oflight may be used in the subject methods for application of thestimulus. Light sources suitable for use in the subject methods include,but are not limited to, UV lamps (e.g., a xenon flash lamp) and laserlight sources (e.g., ultraviolet lasers) that irradiate light at anappropriate wavelength suitable for absorption by the probe. In certaincases, application of the stimulus occurs via fluorescence resonanceenergy transfer (FRET) from a donor chromophore. Laser light sourcesinclude the frequency-doubled ruby laser, which produces a, e.g., 200 mJpulse at 347 nm in 50 ns, and a nitrogen laser (producing e.g., 200 mJat 337 nm), where sufficient intensity can be achieved by focusing thelight through a microscope objective. Any suitable lasers may beconfigured to produce brief (ns) pulses of monochromatic light ofintensity sufficient to modify a probe in a sample. Xenon flash lampsproduce a broad spectrum, from 250 to 1500 nm, and may produce pulses ofabout 1 ms. Filters may be placed in the light path to narrow thespectrum and remove wavelengths (e.g., <300 nm). In certain cases, afterfiltering, the total output of the lamp may between about 300 and about400 nm (e.g., between about 320 nm to about 380 nm, between about 330 nmto about 370 nm, or between about 340 nm to about 360 nm) can beconfigured to produce between about 50mJ and about 250mJ (e.g., about200 mJ) light of intensity sufficient to modify a probe in a sample.

The light source may have a spectral energy distribution suitable forthe particular photoactive group (e.g., photolabile, photoisomerizableor photocleavable group) being used in conjunction with the probe. Insome cases, photolytic cleavage of a masked group is dependent on thewavelength of the irradiating light, its intensity and duration. Forexample, long-wave UV, i.e., UV-A, which has spectral energy in therange of about 320-400 nanometers (nm), is suitable for cleavingo-nitrobenzyl groups. A bulb providing a light intensity at the samplein the range of about 0.2 to about 10 mW/cm² at 365 nm with a 10 nmbandpass may be suitable for such purposes. Light sources of interestinclude, but are not limited to: chemists' mercury spot lamps with 110watts BL9 phosphorescent bulbs, 100 W xenon arc lamp which is passedthrough Hoya 340 and Schott WG 305 filters before illuminating thesample, one or more flashes (e.g., a 50-ns flash) from afrequency-doubled ruby laser that delivers 347 nm light with an averageenergy of 90 mJ (range 83-104 mJ).

It should be understood that the aforementioned wavelength range may beselected as a compromise between using shorter wavelengths that maydamage components of the sample (e.g., wavelengths below 300 nm) andusing longer wavelengths that may be less effective at, e.g., cleavingthe masked group (e.g., wavelengths above 500 nm). Light having otherspectral energy distributions may be required for cleaving otherphotocleavable linkers. Such other energy distributions are readilyavailable, or can be readily determined using any convenient method.

A variety of methods for supplying uniform illumination, controllingillumination intensity, controlling illumination time, controllingsample temperature, and spatiotemporal control of illumination may beused. As used herein, the terms illumination and irradiation are usedinterchangeably. In some embodiments, the illumination time is about 30sec or more, such as about 1 minute or more, about 2 minutes or more,about 3 minutes or more, about 5 minutes or more, about 10 minutes ormore, about 20 minutes or more, about 30 minutes or more, about 60minutes or more, or even more. In certain embodiments, the illuminationtime includes flash photolysis pulses from a laser of nanosecond,picosecond or femtosecond pulse width. The light source may be directedonto the sample using any convenient method. In some cases, the lightsource is directed via the optical path of a microscope, where the lightcan be controlled spatially (e.g., by focusing the light into a smallspot at a particular location).

Any suitable methods may be used to evaluate the reversal ofmodification of the 2′-modified RNA, including but not limited to,primer extension methods, sequencing methods and analytical methods.Evaluation may include comparing the results obtained before and afterreversal of modification.

Once modification of the 2′-modified RNA (or particular nucleotidesthereof) has been reversed, the RNA may be further analyzed using anyconvenient method. In some cases, the subject methods is traceless,e.g., the RNA can be returned to its natural unmodified state afterreversing modification. In general terms, 2′-modification of RNA rendersit unresponsive to cloning methods, e.g, cloning methods used toconstruct sequencing libraries. As such, reversible modification of theRNA by the subject methods retains the opportunity to perform furtheranalysis and/or sequencing of RNAs of interest, e.g., by reversetranscription polymerase chain reaction (RT-PCR) methods. In addition,probes that include photoreactive groups are amenable to spatiotemporalcontrol of the reversal of modification by control of the application ofthe stimulus (e.g., light).

Detectable Label Reagent

The subject methods may further include a conjugation step where afunctional group of the probe (e.g., a bioorthogonal group substituentof the aryl or heteroaryl ring) may be utilized to further modify the2′-modified RNA with a modifying moiety. Modifying moieties of interestinclude but are not limited to, specific binding moieties (e.g., smallmolecules, biotin, peptides, proteins, etc.), and detectable labels.

In some cases, the method includes contacting the sample with adetectable label reagent under conditions sufficient to produce alabeled 2′-modified RNA. Any convenient methods can be utilized toconjugate a detectable label reagent to the 2′-modified RNA.

As used herein, a “detectable label reagent” refers to an agent thatincludes a detectable label and a functional group capable ofselectively reacting with a probe in a sample. Any convenient functionalgroups may be used in the subject detectable label reagents. In somecases, the functional group is a bioorthogonal group, such as a groupthat is capable of selectively reacting with a compatible functionalgroup under physiological conditions. A variety of bioorthogonalchemistries and functional groups may be utilized in the subjectdetectable label reagents, including but not limited to, Staudingerligation chemistries and groups (e.g., azido and phosphine groups),copper-free click chemistries and groups (e.g., azido and cyclooctynegroups), oxime or hydrazine chemistries (using aldehydes and ketones).Introduction of such a functional group as a substituent of the aryl orheteroaryl ring (A) allows for the 2′-acylated RNA to be furthermodified with a moiety such as a detectable label (e.g., a fluorophoreor a peptide tag) or a solid support. Functional group chemistries andchemical agents of interest include, but are not limited to, Clickchemistry groups and reagents (e.g., as described by Sharpless et al.,(2001), “Click Chemistry: Diverse Chemical Function from a Few GoodReactions”, Angewandte Chemie International Edition 40 (11): 2004-2021),Staudinger ligation groups and reagents (e.g., as described by Bertozziet al., (2000), “Cell Surface Engineering by a Modified StaudingerReaction”, Science 287 (5460): 2007), and other bioconjugation groupsand reagents (e.g., as described by Hermanson, Bioconjugate Techniques,Second Edition, Academic Press, 2008).

As used herein, a “detectable label” generally refers to an identifyingtag that can provide for a detectable signal, e.g., luminescence (e.g.,photoluminescence (e.g., fluorescence, phosphorescence),chemoluminescence (e.g., bioluminescence), microparticle aggregation orformation, radioactivity, immunodetection, enzymatic activity, and thelike.

“Fluorophore” refers to a molecule that, when excited with light havinga selected wavelength, emits light of a different wavelength, which mayemit light immediately or with a delay after excitation. Fluorophores,include, without limitation, fluorescein dyes, e.g.,5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), and2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE); cyanine dyes,e.g. Cy3, CY5, Cy5.5, QUASAR™ dyes etc.; dansyl derivatives; rhodaminedyes e. g. 6-carboxytetramethylrhodamine (TAMRA), CAL FLUOR™ dyes,tetrapropano-6-carboxyrhodamine (ROX). BODIPY fluorophores, ALEXA™ dyes,Oregon Green, pyrene, perylene, benzopyrene, squarine dyes, coumarindyes, luminescent transition metal and lanthanide complexes and thelike. The term fluorophores includes excimers and exciplexes of suchdyes.

Complementary oligonucleotide probes may also be utilized in the subjectmethods, e.g., oligonucleotides that have complementary sequencescapable of selectively hybridizing to a site of interest in the RNA. Insome cases, a complementary oligonucleotide probe may be used inconjunction with a chemical agent stimulus in a templated reaction todirect reversal of modification of the 2′-modified RNA to a desirednucleotide of the RNA. In other cases, a complementary oligonucleotideprobe may be used in conjunction with a modifying moiety such as adetectable label reagent. Any convenient methods may be used to link acomplementary oligonucleotide probe and a chemical agent or modifyingmoiety. Methods of interest include those described by Shibata et al.“Oligonucleotide-Templated Reactions for Sensing Nucleic Acids”,Molecules, 2012, 17, 2446-2463.

Utility

The RNA probes and methods of the invention, e.g., as described above,find use in a variety of applications. Applications of interest include,but are not limited to: research applications and diagnosticapplications. Methods of the invention find use in a variety ofdifferent applications including any convenient application where abiological process may be modulated by the structure of a RNA ofinterest. In such cases, the subject probes and methods may be used toobtain structural data of the RNA of interest that finds use in researchof the biological process, or in the diagnosis of a disease conditionassociated with the biological process.

Also of interest is any application where a RNA target can be reversiblymodified. A variety of RNA targets may be utilized in the subjectmethods, including but not limited to, viral RNA, ribosomal RNA (e.g.,5S or 28S rRNA), messenger RNA, telomerase RNA, aptamers, HOTAIR RNA.

Due to the reversible modification of RNA by the subject probes, themethods described herein can be traceless, e.g., the RNA (or particularnucleotides thereof), can be returned to an unmodified state afterperforming the subject methods. In general terms, 2′-modification of RNArenders the RNA unresponsive to cloning methods, e.g., methods used toconstruct sequencing libraries. As such, the subject methods find use inany application where the opportunity to perform further analysis andsequencing of RNAs is of interest, e.g., after structural data has beenobtained from the RNAs. In addition, probes that include photoreactivegroups find use in applications where spatiotemporal control of thereversal of modification by control of the application of the stimulus(e.g., light), is of interest.

Comparison of In Vitro and In Vivo RNA Structural Data

Aspects of the invention include methods for comparing in vitro and invivo RNA structural data. The subject methods may include: contacting anin vitro sample comprising a first RNA, with a first RNA probe toacylate one or more unconstrained nucleotides of the first RNA toproduce a first 2′-acylated RNA, and analyzing the first 2′-acylated RNAto obtain in vitro structural data; contacting an in vivo samplecomprising a second RNA with a second RNA probe to acylate one or moreunconstrained nucleotides of the second RNA to produce a second2′-acylated RNA in vivo, and analyzing the 2′-acylated RNA to obtain invivo structural data; and comparing the in vitro structural data withthe in vivo structural data.

In some embodiments, the method further comprises identifying one ormore nucleotides of the RNA that are differentially acylated in vitroversus in vivo. The subject methods find use in elucidating differencesin the structure of RNA in vivo versus in vitro. The subject methodsfind use in elucidating sites of the RNA structure that are involved ininteractions with other biomolecules in the sample. In some embodiments,the in vitro sample is a control sample to which a variety of controlcomponents may be added, e.g., biomolecules, or fragments thereof, whichare known to interact which the RNA of interest. In such methods, thecomponents of the control sample may be selected to probe one or moreinteractions of the RNA with biomolecules of interest in the non-controlsample.

The subject methods can be adapted for use in comparing RNA structuraldata between any two first and second samples, to identify differencesbetween the RNA structure and/or function. The first and second samplesmay both be of the same type (e.g., both in vivo samples or both invitro samples). Any two samples of interest can be compared using thesubject methods.

The first and second RNA probes may be independently any convenient RNAprobe (e.g., as described herein). In the subject methods, the first andsecond RNA probes may each be independently described by formula (I):

where A is an aryl or heteroaryl ring; R¹ is H or a lower alkyl; and Yis a hydroxyl-reactive group. In certain instances, Y is an active ester(e.g., an acyl imidazole). In some cases, R¹ further comprises an azidogroup. The aryl or heteroaryl ring A may further comprise a bioorthonalgroup. The bioorthogonal group may be any suitable functional group thatis stable under physiological conditions but which is capable ofreacting with a suitable detectable label reagent. Any convenientchemistries may be utilized to add a detectable label to a 2′-modifiedRNA of interest.

Diagnosis of a Disease Condition

The subject compounds and methods find use in a variety of diagnosticapplications, including but not limited to, the development of clinicaldiagnostics, e.g., in vitro diagnostics or in vivo diagnostics where thestructure and/or function of a RNA of interest is implicated. Suchapplications are useful in diagnosing or confirming diagnosis of adisease condition, or susceptibility thereto, determining the propercourse of treatment for a patient suffering from a disease condition.The methods are also useful for monitoring disease progression and/orresponse to treatment in patients who have been previously diagnosedwith the disease. Diagnostic applications of interest include diagnosisof disease conditions, such as those conditions described above,including but not limited to: cancer, inhibition of angiogenesis andmetastasis, cancer-related pain, metastatic breast cancer, etc.

Any suitable methods may be used to perform the subject methods ofdiagnosing a disease condition. Methods of interest that may be adaptedare necessary for practicing the subject methods include, but are notlimited to, sample extraction and preparation methods, detection andquantification methods, RT-PCR methods, normalization methods, etc.

Aspects of the invention include methods of diagnosing a cellularproliferative disease condition. The subject methods may include:contacting a cell comprising a RNA with a RNA probe under conditionssufficient to acylate one or more unconstrained nucleotides of the RNAto produce a 2′-modified RNA; and evaluating the 2′-modified RNA todiagnose the presence or absence of a cellular proliferative diseasecondition.

In some embodiments, the RNA is HOTAIR RNA. In the subject methods, thefirst and second RNA probes may each be independently described byformula (I):

where A is an aryl or heteroaryl ring; R¹ is H or a lower alkyl; and Yis a hydroxyl-reactive group. In certain embodiments, Y is an activeester (e.g., an acyl imidazole).

In some embodiments, R¹ further comprises an azido group. The aryl orheteroaryl ring A may further comprise a bioorthogonal group (e.g., asdescribed above).

Kits

Aspects of the invention further include kits, where the kits includeone or more components employed in methods of the invention, e.g., RNAprobes, RNAs of interest, buffers, SHAPE analysis reagents,stimulus-applying components, detectable label reagents, and cells, asdescribed herein. In some embodiments, the subject kit includes a RNAprobe (e.g., as described herein) and one or more components selectedfrom a modification buffer, a stimulus-applying component and adetectable label reagent.

A variety of components suitable for use in analyzing RNA (e.g., bySHAPE analysis) may find use in the subject kits. Any of the componentsdescribed herein may be provided in the kits. For example, componentssuitable for use in application of stimuli (i.e., stimulus-applyingcomponents), components suitable for use in structural analysis of RNA,including but not limited to, components for RNA sequencing by reversetranscriptase PCR, components for chemical methods of RNA analysis suchas SHAPE analysis, e.g., buffers, cells, complementary DNA primers,enzymes such as reverse transcriptases, N-methylisotoic anhydride (NMIA)or 1-methyl-7-nitroisatoic anhydride (1M7) probes, etc. The subject kitsmay further comprise additional reagents which are required for orconvenient and/or desirable to include in the reaction mixture preparedduring the subject methods, where such reagents include reagents andbuffers for RNA analysis; reagents for adding a detectable label;columns; and the like. Kits may also include tubes, buffers, etc., andinstructions for use. The various reagent components of the kits may bepresent in separate containers, or some or all of them may bepre-combined into a reagent mixture in a single container, as desired.

In some embodiments, the kit finds use in the reversible modification ofRNA, the kit comprising a reversible probe of the invention; and astimulus-applying component, such as a light source or a reagent forunmasking a masked group of the reversible probe.

The stimulus-applying component may be any suitable component (e.g.,equipment, a chemical or biological agent) that finds use in theapplication of a stimulus to a sample (e.g., irradiation of light orcontact with a chemical agent). In certain cases, the stimulus-applyingcomponent is a UV light source, or a chemical agent. The chemical agentmay be supplied in any convenient form, including but not limited to, alyophilized solid or a solution.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, etc. Yet another form of these instructions isa computer readable medium, e.g., diskette, compact disk (CD), etc., onwhich the information has been recorded. Yet another form of theseinstructions that may be present is a website address which may be usedvia the internet to access the information at a removed site.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric

General Methods and Materials

Although, the following protocols are described for probes NAI and FAI,the protocols can be adapted for use with any suitable probe.

Synthetic Methods. 2-methylnicotinic acid, 2-methyl-3-furoic acid and1,1′-carbonyldiimidazole are purchased from Sigma-Aldrich and used asreceived. Anhydrous dimethylsulfoxide (DMSO) is purchased from AcrosOrganics and used as received. NMR is performed on a Varian 500 MHzinstrument and all spectra referenced to the residual solvent peak.

2-methylnicotinic acid imidizolide (NAI). 137 mg (1 mmol)2-methylnicotinic acid is dissolved in 0.5 mL anhydrous DMSO. A solutionof 162 mg (1 mmol) 1,1′-carbonyldiimidazole in 0.5 mL anhydrous DMSO isadded dropwise over 5 minutes. The resulting solution is stirred at roomtemperature until gas evolution is complete, then stirred at roomtemperature for one hour. The resulting solution is used as a 1.0 Mstock solution (assuming complete conversion) containing a 1:1 mixtureof the desired compound and imidazole. The solution is frozen at −80° C.when not in use. An analytical sample is prepared by use ofdichloromethane as solvent instead of DMSO. The crude reaction ispurified by flash silica column chromatography, eluting with ethylacetate.

¹H NMR (500 MHz, CDCl₃): 2.61 (s, 3H), 7.15 (s, 1H), 7.30 (m, 1H), 7.42(s, 1H), 7.73 (dd, 1H, J=8 Hz, 2 Hz), 7.88 (s, 1H), 8.72 (dd, 1H, J=5Hz, 2 Hz); ¹³C NMR (125 MHz, CDCl₃): 23.0, 117.2, 120.6, 127.8, 131.6,135.9, 137.6, 152.1, 157.1, 165.2; HRMS (Calc M+H=188.0818): 188.0819.

2-methyl-3-furoic acid imidazolide (FAI). 126 mg (1 mmol)2-methyl-3-furoic acid is dissolved in 0.5 mL anhydrous DMSO. A solutionof 162 mg (1 mmol) 1,1′-carbonyldiimidazole in 0.5 mL anhydrous DMSO isadded dropwise over 5 minutes. The resulting solution is stirred at roomtemperature until gas evolution is complete, then further stirred atroom temperature for one hour. The resulting solution is used as a 1.0 Mstock solution (assuming complete conversion) containing a 1:1 mixtureof the desired compound and imidazole. The solution is frozen at −80° C.when not in use. An analytical sample is prepared by use ofdichloromethane as solvent instead of DMSO. The crude reaction ispurified by flash silica column chromatography, eluting with 1:1hexanes:EtOAc. NMR indicates the presence of some hydrolyzed material(identical to the furoic acid starting material).

¹H NMR (500 MHz, CDCl₃): 2.60 (s, 3H), 6.63 (d, 1H, J=2 Hz), 7.16 (s,1H), 7.39 (d, 1H, J=2 Hz), 7.55 (s, 1H), 8.20 (s, 1H); ¹³C NMR (125 MHz,CDCl₃): 13.9, 110.3, 111.1, 117.5, 130.5, 137.5, 140.5, 141.5, 162.3;HRMS (Calc M+Na=199.0478): 199.0481.

Characterization of NAI and FAI Reactivity with ATP

ATP gel shift. Although, the following protocol is described for ATP,the protocols can be adapted for use with any suitable RNA. ATP gelshift reactions are carried out as described by Weeks et al., “RNAstructure analysis at single nucleotide resolution by selective2′-hydroxyl acylation and primer extension (SHAPE).” J. Am. Chem. Soc.127, 4223-4231, (2005). Briefly, 10,000 cpm/uL of radiolabeled ATP isincubated with increasing amounts of NAI or FAI (10% final volume) in100 mM HEPES buffer, pH 8.0, containing 6 mM MgCl₂, 100 mM NaCl.Reactions are stopped by addition of an equal volume of Gel LoadingBuffer 11 (Ambion, Inc.) and placed on ice. Reactions are loaded onto30% native polyacrylamide gels (29:1 acrylamide:bisacrylamide, 1% TBE)and visualized by phosphorimaging (STORM, Molecular Dynamics). Singleadduct reaction rates and percentages are calculated by integratingbands (Image Quant, IM Support) and fit to a single exponential.

Quenching ATP reaction with β-mercaptoethanol (BME). 10,000 cpm/uL ofradiolabeled ATP is either preincubated (+) or not (−) with BME finalconcentration 700 mM in 100 mM HEPES buffer, pH 8.0, containing 6 mMMgCl₂, 100 mM NaCl. Immediately after the addition of BME, NAI or FAI(10% final volume) is added and the solution incubated for 5 minutes atroom temperature. Reactions are stopped by addition of an equal volumeof Gel Loading Buffer 11 (Ambion, Inc.) and placed on ice. Gel imagesare integrated as above.

Quenching of acylation reactions. An optional quench step is performedfor probes having extended reactivity by adapting methods as are usedfor other RNA modification procedures. See e.g., Zaug, A. J. & Cech, T.R. Analysis of the structure of Tetrahymena nuclear RNAs in vivo:telomerase RNA, the self-splicing rRNA intron, and U2 snRNA. RNA 1,363-374 (1995). This step allows the experimenter to terminate thereaction at will and perform precise time-course experiments.

Hydrolysis of NAI and FAI. Hydrolysis of NAI and FAI is monitored byadding 1.5 μL of a 100 mM solution of either NAI or FAI in spectroscopicgrade DMF to 598.5 μL of buffer (100 mM HEPES, 100 mM NaCl, 10 mM MgSO₄,pH 8.0) and monitoring the decrease in absorbance at 265 nm (NAI) or 275nm (FAI). Three parameter pseudo-first order exponential decay kineticsare fit using OriginPro 8.0 software using the equation:

f=y0+a*exp(−b*x)

Compound b Half-Life (Min) FAI .009451 73.34 NAI .020476 33.86 The r²for each fit is greater than 0.999Characterization of NAI and FAI Reactivity with RNA

Acylation of RNA, in vitro. In a typical in vitro modification protocol,6 μg total RNA is heated in metal-free water for two minutes at 95° C.The RNA is then flash-cooled on ice. The RNA 3× SHAPE buffer (333 mMHEPES, pH 8.0, 20 mM MgCl₂, 333 mM NaCl) is added and the RNA allowed toequilibrate at 37° C. for ten minutes. To this mixture, 1 μL of 10×electrophile stock in DMSO (+) or DMSO (−) is added. The reaction ispermitted to continue until the desired time. Reactions are quenchedwith 60 μL of DMS stop solution (0.5 M 3-mercaptoethanol, 0.75 MNa.acetate, pH 5.5). Reactions are extracted once with acidphenol:chloroform (pH 4.5±0.2) and twice with chloroform. RNA isprecipitated with 40 μL of 3M sodium acetate buffer (pH 5.2) and 1 μL ofglycogen (20 ug/uL). Pellets are washed twice with 70% ethanol andresuspended in 10 μL RNase-free water.

Acylation of RNA in mouse embryonic stem cells. Although, the followingprotocol is described for mouse embryonic stem cells, the protocol canbe adapted for use with any suitable cells. V6.5 mouse embryonic stemcells are grown under feeder-free conditions as described by Niwa et al.(“Quantitative expression of Oct-3/4 defines differentiation,dedifferentiation or self-renewal of ES cells.” Nat. Genet. 24, 372-376,2000) and Zhang et al. (“Post-translational modification of POU domaintranscription factor Oct-4 by SUMO-1.” FASEB J 21, 3042-3051, 2007).Cells are washed 3× with PBS, then scraped and spun down at 700 rpm for5 minutes. Cells (˜3-6×10⁷) are resuspended in PBS, and DMSO (−), 10%final concentration, or electrophile in DMSO (+) is added to the desiredfinal concentration. Cells suspensions are placed at 37° C. and reactedfor the desired time. Reactions are terminated with β-mercaptoethanol at0.7 M final concentration and reacted at 37° C. for an additional 5minutes. Cells are then spun down and decanted. To the pelleted cells, 1mL of trizol LS (Ambion, Inc.) is added, followed by 200 uL ofchloroform. RNA is precipitated following the trizol LS manufacturer'sinstructions. RNA is resuspended to a concentration of 3 ug/10 uL.Reverse transcription primer used for mouse 5S rRNA:5′-AAAGCCTACAGCACCCGGTAT (SEQ ID NO:1).

Acylation of RNA in human MDA-MB-231 cells. Although, the followingprotocol is described for human MDA-MB-231 cells, the protocol can beadapted for use with any suitable cells. MDA-MB-231 cells are grown inD-MEM (high glucose) culture medium supplemented with 10% fetal bovineserum (FBS) 0.1 mM MEM NonEssential Amino Acids (NEAA), 2 mML-glutamine, 1% Pen-Strep. Cells are washed 3× with PBS, then scrapedand spun down at 700 rpm for 5 minutes. Cells (˜3-6×10⁷) are resuspendedin PBS, and DMSO (−), 10% final concentration, or electrophile in DMSO(+) is added to the desired final concentration. Cells suspensions areplaced at 37° C. and reacted for the desired time. Reactions areterminated with 6-mercaptoethanol at 0.7 M final concentration andreacted at 37° C. for an additional 5 minutes. Cells are then spun downand decanted. To the pelleted cells, 1 mL of trizol LS (Ambion, Inc.) isadded, followed by 200 uL of chloroform. RNA is precipitated followingthe trizol LS manufacturer's instructions. Pellets are washed twice with70% ethanol and resuspended in 10 uL RNase-free water. Reversetranscription primer used for human 5S rRNA: 5′-aaagcctacagcacccggtat(SEQ ID NO:2).

Acylation of RNA in yeast cells. Although, the following protocol isdescribed for yeast cells, the protocol can be adapted for use with anysuitable cells. Yeast cells are grown to an OD₆₀₀ of 1.0 at 30° C. inYPD medium. Cells are spun down at 4000×g and decanted. Cells areresuspended in PBS, and DMSO (−), 10% final concentration, orelectrophile in DMSO (+) is added to the desired final concentration.Cell suspensions are placed at 37° C. and reacted for the desired time.Reactions are terminated with 6-mercaptoethanol at 0.7 M finalconcentration and reacted at 37° C. for an additional 5 minutes. Cellsare then spun down, decanted, and flash frozen in liquid nitrogen.Frozen pellets are resuspended in 500 uL of 50 mM NaOAc pH5.0, 10 mMEDTA pH 8.0 and 100 uL of 10% SDS. To the mixture 700 uL of saturatedphenol is added. Cells are incubated at 65° C. for 1 minute. The freezethaw cycle is repeated three times. The aqueous phase is separated withPCI (phenol:chloroform:isoamyl alcohol=50:48:2. The aqueous phase isextracted twice with chloroform and added to 3 volumes of ethanol andone-tenth volume of 3M NaOAc pH 5.0. Pellets are washed twice with 70%ethanol and resuspended in 10 uL RNase-free water. Reverse transcriptionprimer used for yeast 5S rRNA: 5′-AGATTGCAGCACCTGAGTTT (SEQ ID NO:3)

Acylation of RNA in E. coli cells. Although, the following protocol isdescribed for E. coli cells, the protocol can be adapted for use withany suitable cells. E. coli cells are grown to an OD₆₀₀ of 0.25 at 37°C. in YPD medium. Cells are spun down at 4000×g and decanted. Cells areresuspended in PBS, and DMSO (−), 10% final concentration, orelectrophile in DMSO (+) is added to the desired final concentration.Cells suspensions are placed at 37° C. and reacted for the desired time.Reactions are terminated with β-mercaptoethanol at 0.7 M finalconcentration and reacted at 37° C. for an additional 5 minutes. Cellsare then spun down, decanted, and flash frozen in liquid nitrogen. Cellsare resuspended in a final volume of a fresh solution of 800 μl 0.5mg/ml lysozyme, TE pH 8.0. 80 μl of 10% SDS is added to the mixture andthe slurry is placed at 64° C. for 1-2 min. After incubation add 88 μl 1M NaOAc, pH5.2 is added. The samples are added to an equal volume (1 ml)of water-saturated phenol (pH<7.0) and incubated at 64° C. for 6 min.The resultant slurry is spun at max speed (14,000 rpm) for 10 min at 4°C. The aqueous phase is extracted twice with chloroform and added to 3volumes of ethanol and one-tenth volume of 3M NaOAc pH 5.0. Pellets arewashed twice with 70% ethanol and resuspended in 10 μL RNase-free water.Reverse transcription primer used for E. coli 5S rRNA:5′-TGCCTGGCAGTTCCCTACTC (SEQ ID NO:4)

Acylation of RNA in Drosophila S2 cells. Although, the followingprotocol is described for Drosophila S2 cells, the protocol can beadapted for use with any suitable cells. Drosophila S2 cells are grownat 25° C. in Schneider's Drosophila Medium (Invitrogen, Carlsbad,Calif.) supplemented with 10% Fetal Bovine serum (SAFC Biosciences,Lenexa, Kans.) and Penicillin-Streptomycin (Invitrogen, Carlsbad,Calif.). Cells are washed 3× with PBS, then scraped and spun down at 700rpm for 5 minutes. Cells (˜3-6×10⁷) are resuspended in PBS, and DMSO(−), 10% final concentration, or electrophile in DMSO (+) is added tothe desired final concentration. Cells suspensions are placed at 37° C.and reacted for the desired time. Reactions are terminated withβ-mercaptoethanol at 0.7 M final concentration and reacted at 37° C. foran additional 5 minutes. Cells are then spun down and decanted. To thepelleted cells, 1 mL of trizol LS (Ambion, Inc.) is added, followed by200 μL of chloroform. RNA is precipitated following the trizol LSmanufacturer's instructions. Pellets are washed twice with 70% ethanoland resuspended in 10 μL RNase-free water. Reverse transcription primerused for Drosophila 5S rRNA: 5′-CGAGGCCAACAACACGCGGT (SEQ ID NO:5)

Acylation and enrichment for nuclear RNAs in HeLa S3 cells. Although,the following protocol is described for HeLa S3 cells, the protocol canbe adapted for use with any suitable cells. HeLa S3 cells are grown inD-MEM (high glucose) culture medium supplemented with 10% fetal bovineserum (FBS) 0.1 mM MEM NonEssential Amino Acids (NEAA), 2 mML-glutamine, 1% Pen-Strep. Cells are washed 3× with PBS, then scrapedand spun down at 700 rpm for 5 minutes. Cells (˜3-6×10⁷) are resuspendedin PBS, and DMSO (−), 10% final concentration, or 2M electrophile (200mM final) in DMSO (+) is added to the desired final concentration. Cellsuspensions are placed at 37° C. and reacted for thirty minutes.Reactions are terminated with β-mercaptoethanol at 0.7 M finalconcentration and reacted at 37° C. for an additional 5 minutes. Cellsare then spun down and decanted. HeLa cell pellets are resuspended in 2ml PBS, 2 ml nuclear isolation buffer (1.28 M sucrose; 40 mM Tris-HCl pH7.5; 20 mM MgCl2; 4% Triton X-100), and 6 ml water on ice for 20 min(with frequent mixing). Nuclei are pelleted by centrifugation at 2,500 Gfor 15 min. Nuclear pellet is resuspended in 1 ml RIP buffer (150 mMKCl, 25 mM Tris pH 7.4, 0.5 mM DTT, 0.5% NP40, 1 mM PMSF). Resuspendednuclei are mechanically sheared using a dounce homogenizer with 15-20strokes. Nuclear membrane and debris are pelleted by centrifugation at13,000 RPM for 10 min. To the mixture 700 uL of saturated phenol isadded. Cells are incubated at 65° C. for 10 minutes. The aqueous phaseis separated with PCI (phenol:chloroform:isoamyl alcohol=50:48:2). Theaqueous phase is extracted twice with chloroform and added to 3 volumesof ethanol and one-tenth volume of 3M NaOAc pH 5.0. Pellets are washedtwice with 70% ethanol and resuspended in 10 uL RNase-free water. Homosapiens small nucleolar RNA, C/D box 3A (SNORD3A), RT Primer:ACCACTCAGACCGCGTTCTCTCCC (SEQ ID NO:6). Homo sapiens RNA, U1 smallnuclear 1 (RNU1-1), small nuclear RNA TR Primer:CAGGGGAAAGCGCGAACGCAGTCC (SEQ ID NO:7). Homo sapiens RNA, U2 smallnuclear 1 (RNU2-1), small nuclear RNA RT Primer: GGGTGCACCGTTCCTGGAGG(SEQ ID NO:8).

Reverse Transcription of modified RNA (in vivo and in vitro).³²P-end-labeled DNA primers are annealed to 3 μg of total RNA byincubating at 95° C. for two minutes followed by a step-down cooling (2deg/sec) to 4° C. To the reaction first-strand buffer, DTT and dNTPs areadded. The reaction is pre-incubated at 52° C. for one minute, thensuperscript III (2 units/4 final concentration) is added. Extensions areperformed for ten minutes. To the reaction, 1 μL of 4 M NaOH is addedand allowed to react for 5 minutes. 10 μL of Gel Loading Buffer II(Ambion, Inc.) is then added, and cDNA extensions are resolved on 8%denaturing (7 M Urea) polyacrylamide gels (29:1acrylamide:bisacrylamide, 1% TBE).

Characterization of reverse transcription stops. cDNA extensions arevisualized by phosphorimaging (STORM, Molecular Dynamics). cDNA bandsare integrated with SAFA⁴. SHAPE reactivities are normalized to a scalespanning 0 to ˜1.5, where 1.0 is defined as the mean intensity of highlyreactive nucleotides. RNA secondary structures are predicted using mFOLDsoftware.

Results

Screening of acylation electrophiles. Several acylation electrophiles(Table 1) were screened for selective reactivity toward hydroxyl groups,solubility at high concentrations, and amenability to RNA modificationinside living cells within a reasonable time frame. Qualitative resultsare shown in Table 1.

Compound Result

Control

Too Reactive, High Hydrolysis

Too Reactive, High Hydrolysis

Low ATP Modification

Too Reactive, High Hydrolysis

Good ATP Modification, Low Solubility

Weak ATP Modification.

Unreactive, Low Solubility

Low ATP Modification

Low ATP Modification

Evaluation of exemplary probes NAI and FAI. Both NAI and FAI werereactive with ATP as illustrated in FIG. 3 using methods as describedabove. Both NAI and FAI were found to retain reactivity with ATP at 120minutes in aqueous buffer, whereas NMIA is mostly quenched after thirtyminutes (see Weeks et al., J. Am. Chem. Soc. 127, 4223-4231, 2005). Therate of hydrolysis was evaluated using methods described herein and itwas observed that NAI (t_(1/2) hydrolysis=33 min) and FAI (t_(1/2)hydrolysis=73 min) are considerably more stable in aqueous solution incomparison to NMIA (t_(1/2) hydrolysis=4 min, see Weeks et al.).

Quenching of acylation reaction. Addition of beta-mercaptoethanol (B-Me)according to the method described above halted the gel shift acylationreaction as illustrated in FIG. 7.

Secondary structure of human 5S rRNA in vitro. 5S rRNA was selectedbecause of its abundant nature, highly characterized structure, andability to fold into a stable structure without the need for proteincofactors. Similar quantitative patterns of 2′-hydroxyl acylation withNAI or FAI versus NMIA were observed (e.g., R²=0.93, FIG. 1D and FIG.8). The reactivities of the probes were mapped to the predictedsecondary structure of human 5S rRNA. Modifications by NMIA, FAI and NAIall map to residues that are predicted to be flexible (FIG. 1B and FIG.8). These data suggest that both NAI and FAI are suitable electrophilesfor 2′-hydroxyl acylation on structured RNA molecules, yielding accuratestructural information.

Probing RNA in Live Cells

The ability of NAI and FAI to monitor RNA structure in live cells wasevaluated. Cultured mouse embryonic stem cells (ESC) were reacted withNAI or FAI in aqueous buffer, using methods described above. At 13 mMprobe (the maximum solubility of NMIA), no 5S rRNA modification wasdetected after one hour. At 20 mM probe, positive signals formodification were observed. Both NAI and FAI employed in the cellularexperiments caused blocks in subsequent reverse transcription,suggesting modification, while NMIA did not (FIG. 9). In addition, NAIshowed greater extent of modification than FAI, consistent with itshigher reactivity toward ATP in vitro.

FIG. 9: Characterization of electrophile reactivity with RNA in cells.(A) Increasing amounts of NAI yield concentration-dependent reversetranscription stops. (B) Higher concentrations of NAI and FAI producereverse transcription stops. At the solubility limit of NMIA there is nosign of modification, even on 5S rRNA, one of the most abundanttranscripts in the cell. Notably, NAI gives higher intensity RT stops atthe same concentration as FAI, which is consistent with its higherreactivity to ATP.

NAI probing of murine ESC. Application of 100 mM NAI to murine ESCsaccording to the methods described above, resulted in 5S rRNAmodification in as little as one minute with suitable signal-to-noiseratio. This signal begins to plateau by 15 minutes (FIG. 10). Even after30 minutes of NAI treatment, ESCs remained attached to tissue culturevessel, appeared morphologically normal and unstained by trypan blue(FIG. 10).

To test the ability of the probe reagent to modify lower abundant RNAs,the SHAPE pattern for three nuclear localized RNAs was determined.Significant RNA modification was detected which suggests that thereagent is able to enter the nucleus and react with lower abundant RNAsto give structural information (FIG. 11). NAI also modifies 5S rRNA incultured human cancer cells, Drosophila S2 cells, yeast cells, and E.coli cells, suggesting that it is a general cell-permeable probe of RNAstructure.

The pattern of 5S rRNA SHAPE in ESCs was compared to the crystalstructure of the 80S ribosome from yeast, which includes the 5S rRNA(Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0 Aresolution. Science 334, 1524-152, 2011). Yeast and mammalian 5S rRNAexhibit very high sequence similarity and functional domainarchitecture, as indicated by a CLUSTALW alignment score of 60. Thecrystal structure of the ribosome is validated by decades of moleculargenetics and biochemical studies and likely represents a conformationthat occurs in vivo. Overlaying the SHAPE data with the subject probesto the 5S crystal structure showed that practically all residues inflexible regions or not in canonical Watson-Crick base-pairs aremodified, including single-stranded loops, unstable non-canonicalbase-pairs, and a single base flipped out of the helical duplex (FIG.2). These results indicate that the subject reagents probe RNA structurein vivo with high accuracy and single-nucleotide resolution.

Comparison of SHAPE profiles of 5S rRNA in vivo versus in vitro revealkey RNA-RNA and RNA-protein interactions that dock the 5S rRNA into theribosome. Overall, the profiles look similar, but a few key differencessuggest differential interactions in the living system (FIG. 3, A andB). FIG. 3 illustrates that 5S rRNA has different modification patternsin cells: (A) Denaturing gel electrophoretic analysis of NAImodification of 5S rRNA in M. musculus Embyronic Stem cells and invitro; and (B) Normalized Differential profile of M. musculus EmbyronicStem cell 5S rRNA.

Hereafter, residues in 5S are numbered per the mouse gene (M. musculus,M.M.); residues in other ribosomal subunits are numbered as in the yeastcrystal structure (S. cerevisiae, S.C.). First, differences between thein vitro and in vivo modification profiles were observed with residueM.M.A49/M.M.A50. Within the context of the crystal structure it is notedthat the analogous residue S.C.U51, which sits near the nexus of Loop Band Helix III, is kinked to allow the docking of Loop C into the 28SrRNA (Resi. S.C.C2684 and S.C.U2683). This conformation permits theresidues S.C.ARG218, S.C.LEU222, S.C.GLU221, and S.C.LYS224 to bestacked against residues S.C.A51 and S.C.U50 (FIG. 4D). As a result,S.C.U51 seems to be pushed out of the helix, thus increasing its dynamicnature and exposing the 2′-OH for reactivity. Prior saturationmutagenesis showed that S.C.A51 and S.C.U50 make contact with ribosomalproteins S.C.L11 and S.C.L5 to form a critical structural link betweenthe large and small ribosomal subunits and are essential for properribosome function and viability (Smith et al., Saturation mutagenesis of5S rRNA in Saccharomyces cerevisiae. Mol Cell Biol 21, 8264-827, 2001;Yusupov et al. Crystal structure of the ribosome at 5.5 A resolution.Science 292, 883-896, 2001). Thus, the subject probes can read outalterations in the RNA tertiary structure as a result of samplingcritical mature ribosome conformations.

Second, a three-nucleotide bridge that connects Helix II with Loop A(M.M.C10, M.M.U11, and M.M.G67) showed significant differences (FIG.4E). Within the crystal structure S.C.C10, S.C.A11, S.C.G67 are engagedin a three-nucleotide bridge. S.C.G67 and S.C.A11 are involved inextensive hydrogen bonding interactions that may stabilize them inlower-reactive conformations. S.C.C10 also moves out of the helix tostack on S.C.PHE20. This conformation exposes the 2′-OH of S.C.C10 andmay result in increased reactivity to NAI in vivo.

Third, the residues of M.M.U84/M.M.A83, which are in Loop D, were morereactive to NAI in vitro. Within the context of the 80S ribosome theseresidues are engaged in extensive hydrogen-bond contacts with residuesS.C.G1148 and S.C.G1171 of 28S S.C.rRNA. S.C.U86 is stacked uponS.C.A1197 and is in an H-bonding contact with cobalt hexamine (FIG. 4F).Notably, mutations of these 5S residues in yeast result in gross defectsin translational accuracy. These differences suggest that NAI is able todistinguish subtle dynamic differences that may be the result of proteininteractions, yet can still identify residues that are unpaired andtherefore more flexible in the context of the cell. These findingssuggest in vivo versus in vitro SHAPE comparison as a powerful unbiasedstrategy to pinpoint key residues in ncRNA interaction and function.

Comparison of the predicted RNA secondary structure and the present datashowed that the subject reagent was unable to produce significantmodification patterns from residues in Loop E in the M.M. 5S pattern.Existing reagents also failed to probe Loop E in vitro, and its highdegree of hydrogen bonding and stacking interactions that are thought toprevent Loop E residues from interacting with single-strand specificreagents and RNases. Loop E was slightly more reactive to NAI in vitro,and these residues are completely devoid of modification in vivo. Thisresult is surprising because the S.C. crystal structure showed tworesidues, C72 and C73, to be pushed out of Loop E. These residues alsohave the highest b-factor, suggesting these residues are highly dynamic(FIG. 5G). Analysis of in vitro and in vivo modified 5S rRNA in yeastrevealed several residues with similar modification patterns as in themouse 5S rRNA. Further, residues with lower b-factors in the crystalstructure were shown to be less reactive in vivo. Importantly, residuesC72 and C73 displayed the largest differences in the cell, with a markedincrease in reactivity (FIG. 5, H and I). Moreover, residues withaltered modification pattern in vivo are often required for 5S functionin vivo when mutated (FIG. 51).

These results show that the subject probes are able to distinguishfunctional RNA dynamics that are the result of a mature cellularcomplex, and can be used to compare X-ray structures to interrogatecellular RNA complexes. This analysis is the first comparison of 5S rRNAstructure in vitro versus in vivo. Overall, these experiments establishthat the subject acylation reagents are capable of modifying RNA in vivoand can sensitively read characteristics of RNA structure that are theresult of unique conformations that RNA adopts in the cell.

Modification of cellular RNA from several species. FIG. 12 illustratesthe results of acylation reactions using NAI with denaturing gelelectrophoresis: (A) Homo sapiens MDA-MB-231 cells 5SrRNA RT products;(B) Saccharomyces cerevisiae cells 5S rRNA RT products; (C) Escherichiacoli cells 5S rRNA RT products; (D) Drosophila melanogaster cells 5SrRNA RT products.

Synthesis and evaluation of azido containing probes. Azido containingprobes are prepared according to the synthetic scheme shown in FIG. 13B.ABI-1 (FIG. 13B) was prepared starting with commercially availablemethyl 2-methylbenzoate. The compounds are screened to optimize twoparameters: acylation time and solubility. ABI-1 satisfied thesecriteria with medium solubility (20 mM, 10% DMSO) and similar kineticsof acylation of ATP as compared to the anhydride reagent NMIA (FIG.13C). These data show that ABI-1 is a suitable electrophile for2′-hydroxyl acylation.

The isolated ATP-(ABI-1) adducts were incubated with a series oftriarylphosphine compounds at room temperature. ATP was quantitativelydeacylated in as little as 15 minutes and NMIA, a negative control, wasnot (FIG. 13D). ABI-1 probes are removed from RNA without compromisingthe integrity of the RNA itself. Together, these results demonstrate thefeasibility of the catch-and-release RNA modification protocol.

ABI-1 as a probe of RNA secondary structure. To evaluate the compound'sability to modify RNA at conformationally flexible positions ABI-1 wastested in comparison to NMIA. ABI-1 and NMIA were incubated with invitro transcribed RNA and with DMSO as a negative control. Afterincubation, the RNA was extracted and precipitated with ethanol. Theresuspended RNA was then reverse transcribed with a radiolabeled primerand cDNA products were analyzed on a sequencing gel (FIG. 14A). As shownin FIG. 14, A and B there is little difference between the cDNA productscreated from ABI-1 treated RNA and RNA exposed to NMIA. Both reagentssuccessfully mapped the predicted secondary structure of the 5′ end ofHOTAIR, a long non-coding RNA. Overall, these results establish thereversible modification strategy to accurately measure RNA secondarystructure.

ABI-1 Reversible Modification and Cell-Permeability. The ability ofABI-1 to reversibly acylate RNA, without compromising the integrity ofthe full-length transcript was investigated. ABI-1 is incubated with invitro transcribed RNA (+) and with DMSO as a negative control (−). Afterincubation the RNA is extracted and precipitated with ethanol. The RNAis then subjected to deprotection conditions (80° C., 20 minutes and 16mM triphenylphosphine (TPP)). The RNA is then precipitated and theresuspended RNA is then reverse transcribed with a radiolabeled primerand cDNA products are analyzed on a sequencing gel (FIG. 15, A). Theresults showed that in the presence of TPP the RNA is deprotected, (seee.g., FIG. 13D), without compromising the integrity of the RNA itself.Nicotinic acid derivative NAI-1 was evaluated for permeating cellmembranes and reading out RNA secondary structure accurately (see FIG.15C). The data shows that NAI-1 was able to read RNA secondary structureand permeate cells._FIG. 15A. Demonstration of RNA deprotection withABI-1; FIG. 15B. Chemical structures of ABI, NAI, and NAI-1; FIG. 15C.Demonstration of cell-permeability and structural modification of RNA incells using NAI-1.

Tagging Acylation Chemicals Through “Click” Chemistry (FIG. 16). Thecompound NAI-1 was used for conjugation to both fluorophores andenrichment handles using “click” chemistry. Using the azide-linkedacylation reagent we have been able to link fluorescent dye to NAI-1/RNAcomplexes. We used DIBO-click for copper-free chemistry to conjugate toRNA (Summarized in FIG. 16, A and B). These results suggest that thesereagents can be conjugated to fluorescent markers for RNA labeling insolution. We also have been able to demonstrate RNA conjugation to asolid-phase resin, as is shown by depletion of click-fluorophore signal(FIG. 16, B; lane 3 and 4).

We have also demonstrated that NAI-1 can be used to tag RNAs with anenrichment handle and that this tagging is dependent on the azideacylation handle. As shown in FIG. 16, C we can acylate hydroxyls fromATP (an acylation mimic of RNA). We can also supershift the acylated ATPwith copper free “click” chemistry with DIBO-biotin. The scheme isoutlined in FIG. 16, D. We were also able to show that we can isolatebiotin-conjugated RNAs using a pulldown. We can show enrichment forbiotin labeled RNAs using a streptavidin pulldown in which the isolatedRNAs are eluted then probed using a streptavidin dot-blot (FIG. 16, E).Together these results demonstrate that NAI-1, or other azidefunctionalized acylation chemicals can be functionalized after theirconjugation to RNA.

Enriching for Acylated RNAs to Decrease Background in RNA StructureProbing Experiments. Using conditions discussed in FIG. 16, we canenrich for RNAs that have been acylated with NAI-1. We then tested theability of this enrichment strategy to isolate RNA structure signaturesassociated with acylation.

In a given RNA structure probing experiment the amount of RNA that isacylated under single-hit kinetics is ˜15-20% of the total RNA. This canoften result in a substantial amount of background that results fromeither spurious RT stops or full-length cDNAs that arise fromreverse-transcription on unmodified RNAs. As shown in FIG. 17, we havebeen able to isolated purified RNAs (IP lane) that map back to segmentsof RNA we predict to be acylated (single-stranded regions, denoted withorange circles). We also de-enrich for spurious stops that map to knowndouble stranded regions (double-stranded regions, denoted with bluecircles). This enrichment is also dependent on our acylated RNAs beingfunctionalized with biotin through our copper-free “click” chemistry(−biotin vs +biotin; FIG. 17, A). We also significantly de-enrich forfull-length cDNAs; the full-length band for un-modified cDNAs isoutlined in red. Overall, this data suggest were are able to isolateacylated RNAs and wash away RNAs that are unmodified, furtherdemonstrating the feasibility of labeling and manipulating RNA withthrough our novel reagents.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method for obtaining structural data from a RNA in a sample, themethod comprising: contacting the sample with a SHAPE probe to acylateone or more unconstrained nucleotides of the RNA to produce a2′-acylated RNA, wherein the SHAPE probe is described by formula (II):

wherein: A is an aryl or a heteroaryl ring; R¹ is H or a lower alkyl; R²is H or a lower alkyl; and analyzing the 2′-acylated RNA to obtainstructural data.
 2. The method of claim 1, wherein A comprises one ormore groups independently selected from a solubility-enhancing group, abinding moiety, a tag, a permeability-enhancing group, a detectablelabel and a bioorthogonal group.
 3. The method of claim 1, wherein theSHAPE probe is described by one of formulas (II) and (III):

wherein: Z¹, Z², Z³, Z⁴ Z⁵, Z⁶ and Z⁷ are independently selected from O,S, CR³, N and NR³, wherein R is H or an aryl group substituent.
 4. Themethod of claim 3, wherein A is selected from a phenyl, a pyridyl,pyrrolyl, a furanyl, a thienyl, a thiazolyl, an imidazolyl, oxazolyl, apyrimidinyl, a pyrazinyl and a pyridazinyl ring.
 5. The method of claim4, wherein the SHAPE probe is described by one of the followingstructures:

wherein Z⁸ is O, S or NR⁴, wherein R⁴ is H or a lower alkyl; and whereinthe imidaolyl group is optionally substituted with a lower alkyl, a haloor nitro.
 6. The method of claim 5, wherein R¹ is a lower alkyl.
 7. Themethod of claim 6, wherein R¹ is selected from one of the followinggroups:

wherein X is selected from —N₃, hydroxyl, protected hydroxyl, protectedthiol, amino and protected amino; and R⁵ is H or an alkyl groupsubstituent.
 8. The method of claim 7, further comprising applying astimulus to the sample to de-acylate the 2′-acylated RNA.
 9. The methodof claim 8, wherein the stimulus is a photon, a deprotection reagent oran azido-reactive agent.
 10. The method of claim 9, wherein: R¹ is—CH—N₃ or —CH₂CH₂N₃; and the stimulus is selected from an arylphosphine,an alkylphosphine, an arylalkylphosphine and a dithiol.
 11. The methodof claim 2, further comprising: contacting the sample with a modifyingreagent under conditions sufficient to conjugate the reagent to afunctional group of the A ring of a 2′-acylated RNA.
 12. The method ofclaim 11, wherein the modifying reagent is selected from a tag (e.g., afluorophore or peptide tag) and a solid support.
 13. The method of claim1, wherein the sample is a cell.
 14. The method of claim 1, wherein thesample is in vivo.
 15. The method of claim 1, wherein the sample is invitro.
 16. A method for reversibly modifying RNA in a sample, the methodcomprising: contacting the sample with a reversible probe to modify oneor more unconstrained nucleotides of the RNA to produce a 2′-modifiedRNA, wherein the probe is described by formula (V):

wherein: Y is a hydroxy-reactive group; A is an aryl or a heteroarylring; and L is a lower alkyl group; analyzing the 2′-modified RNA toobtain structural data; and contacting the sample with an azido-reactivereagent to reverse modification of the 2′-modified RNA.
 17. The methodof claim 16, wherein Y is an active ester and the 2′-modified RNA is2′-acylated RNA.
 18. The method of claim 17, wherein the azido-reactivereagent is selected from an arylphosphine, alkylphosphine, anarylalkylphosphine and a dithiol.
 19. The method of claim 18, whereinthe azido-reactive reagent comprises a binding moiety that specificallybinds to the 2′-acylated RNA adjacent to the site of 2′-acylation (e.g.,oligonucleotide having a sequence that hybridizes to a region of the2′-acylated RNA).
 20. The method of claim 16, wherein A comprises one ormore groups independently selected from a solubility-enhancing group, abinding moiety a permeability-enhancing group and a functional group(e.g., for attaching a tag (e.g., a fluorophore) or a solid support).21. The method of claim 17, wherein the reversible probe is described byone of formulas (VI) and (VII):

wherein: Z¹, Z², Z³, Z⁵, Z⁶ and Z⁷ are independently selected from O, S,CR, N and NR, wherein R is H or an aryl group substituent; X is halo,imidazolyl, an N-hydroxylsuccinimidyl, alkoxy or an aryloxy; and m is 1or
 2. 22. The method of claim 21, wherein A is selected from a phenyl, apyridyl, a pyrrolyl, a furanyl, a thienyl, a thiazolyl, imidazolyl, anoxazolyl, a pyrimidinyl, a pyrazinyl and a pyridazinyl ring.
 23. Themethod of claim 22, wherein the reversible probe is described by one ofthe following structures:

wherein: Z⁸ is O, S or NR⁴, wherein R⁴ is H or a lower alkyl; and m is 1or
 2. 24. The method of claim 23, wherein X is imidazolyl, optionallysubstituted with a lower alkyl, a halo or nitro.
 25. The method of claim24, wherein m is
 1. 26. The method of claim 16, further comprising:contacting the sample with a modifying reagent under conditionssufficient to conjugate the reagent to a functional group of the A ringof a 2′-modified RNA.
 27. The method of claim 26, wherein the modifyingreagent is selected from a detectable label (e.g., a fluorophore orpeptide tag) and a solid support.
 28. The method of claim 16, whereinthe sample is a cell.
 29. The method of claim 28, wherein the sample isin vivo.
 30. The method of claim 16, wherein the sample is in vitro. 31.A method for comparing in vitro and in vivo RNA structural data, themethod comprising: contacting an in vitro sample comprising a first RNA,with a first RNA probe to acylate one or more unconstrained nucleotidesof the first RNA to produce a first 2′-acylated RNA, and analyzing thefirst 2′-acylated RNA to obtain in vitro structural data; contacting anin vivo sample comprising a second RNA with a second RNA probe toacylate one or more unconstrained nucleotides of the second RNA toproduce a second 2′-acylated RNA in vivo, and analyzing the 2′-acylatedRNA to obtain in vivo structural data; comparing the in vitro structuraldata with the in vivo structural data.
 32. The method of claim 31,further comprising identifying one or more nucleotides of the RNA thatare differentially acylated in vitro versus in vivo.
 33. The method ofclaim 31, wherein the first and second RNA probes are each independentlydescribed by formula (I):

wherein: A is an aryl or heteroaryl ring; R¹ is H or a lower alkyl; andY is a hydroxyl-reactive group.
 34. The method of claim 33, wherein Y isan acyl imidazole.
 35. The method of claim 33, wherein R¹ furthercomprises an azido group.
 36. The method of claim 33, wherein A furthercomprises a bioorthonal group. 37-49. (canceled)